Apoptosis
Apoptosis, or programmed cell death, is of fundamental importance to normal biological processes including embryogenesis, maintenance of tissue homeostasis, cellular development of multicellular organisms, elimination of virus-infected cells, and the development of the immune system (Ellis et al., 1991). It is a type of cell death that is fundamentally distinct from degenerative death or necrosis in that it is an active process of gene-directed cellular self-destruction which, in some instances, serves a biologically meaningful homeostatic function.
p53
The p53 protein, originally identified as a tumor-associated antigen, is the product of a tumor suppressor gene that functions to arrest the growth of mutated or aberrant cells (Baker et al, 1990). Functional p53 is believed to detect DNA damage (Lee et al, 1995) and subsequently induce DNA repair (Kastan et al, 1991), growth arrest (Kuerbitz et al, 1992), or apoptosis (Yonish-Rouach et al, 1991) of the aberrant cells. In particular, p53 controls genomic stability by eliminating genetically damaged cells from the cell population, and one of its major functions is to prevent tumor formation.
The p53 protein has at least two DNA-binding sites:
(1) the core of the p53 protein, which interacts specifically with a DNA sequence in the promoter region of p53 responsive genes (el-Deiry et al, 1992); and
(2) the C-terminus of the p53 protein, which can recognize features common to damaged DNA in general (Lee et al, 1995; Foord et al, 1991).
The p53 protein is a transcription factor that binds specifically to a consensus site present in the regulatory sequences of p53-dependent genes (el-Deiry et al, 1992). Mutation of the p53 gene in the domain encoding sequences involved in binding to the specific DNA regulatory site causes a loss of tumor suppression. Therefore, it is not surprising that a significant proportion of natural human tumors bear mutated p53 (Hollstein et al, 1991).
p53 has a short half-life, and, accordingly, is continuously synthesized and degraded in the cell. However, when a cell is subjected to stress, p53 is stabilized. Examples of cell stress that induce p53 stabilization are                a) DNA damage, such as damage caused by UV (ultraviolet) radiation, cell mutations, chemotherapy, and radiation therapy;        b) hyperthermia;        c) hypoxia; and        d) deregulation of microtubules caused by some chemotherapeutic drugs, e.g., treatment using taxol or Vinca alkaloids.        
Stress-activated p53 induces a cascade of events that result in growth arrest or apoptosis of the stressed cell, thereby preventing the outgrowth of aberrant cells and tumor formation (Ko, 1996). However, excessive activation of p53 after severe stress can be harmful to the organism, as tissue function may be damaged by excessive apoptosis (Komarova, 2001).
Specifically, radiation therapy and chemotherapy exhibit severe side effects, such as severe damage to the lymphoid and hematopoietic system and intestinal epithelia, which limit the effectiveness of these therapies. Other side effects, like hair loss, also are p53 mediated and further detract from cancer therapies. Therefore, to eliminate or reduce adverse side effects in normal tissues associated with cancer treatment, it would be beneficial to inhibit p53 activity in normal tissue during treatment of p53-deficient tumors, and thereby protect normal tissue.
Inactivation of p53 has been considered an undesirable and unwanted event, and considerable effort has been expended to facilitate cancer treatment by restoring p53 function. However, p53 restoration or imitation causes the above-described problems with respect to damaging normal tissue cells during chemotherapy or radiation therapy. These normal cells are subjected to stress during cancer therapy, which leads the p53 in the cell to cause a programmed death. The cancer treatment then kills both the tumor cells and the normal cells.
U.S. Pat. No. 6,593,353 discloses p53 inhibitors in the treatment of p53-mediated diseases, conditions and injuries.
U.S. Pat. No. 6,420,136 discloses methods for modulating the activity of the p53 protein in cells by the addition of a protein which enhances or inhibits the biochemical activity of p53.
U.S. Pat. No. 6,630,584 discloses a single chain antibody which recognizes an epitope exposed on mutant, but not on wild-type p53 and a DNA molecule encoding the single chain Fv, pharmaceutical compositions comprising the antibody and methods of treatment using the pharmaceutical compositions.
p53 and Stress-Associated Response
The adverse effects of p53 activity on an organism are not limited to cancer therapies. p53 is activated as a consequence of a variety of stresses associated with injuries (e.g., burns) naturally occurring diseases (e.g., hyperthermia associated with fever, and conditions of local hypoxia associated with a blocked blood supply, stroke, and ischemia) and cell aging (e.g., senescence of fibroblasts), as well as a cancer therapy. Temporary p53 inhibition, therefore, also can be therapeutically effective in: (a) reducing or eliminating p53-dependent neuronal death in the central nervous system, i.e., brain and spinal cord injury, (b) the preservation of tissues and organs prior to transplanting, (c) preparation of a host for a bone marrow transplant, and (d) reducing or eliminating neuronal damage during seizures, for example.
In addition, various degenerative diseases, including Alzheimer's disease, Parkinson's disease, ischemic stroke (Mattson, 2001; Martin, 2001), glaucoma (Nickells, 1999) secondary degeneration after trauma (Raghupathi, 2000), myocardial infarction (Haunstetter, 1998) are associated with excessive cell death of sensitive tissue in response to stress. Therefore, temporary inhibition of stress-related cell death may serve the prevention and therapy of degenerative diseases (Komarova, 2001).
Monoclonal Antibody to the DNA-Binding Domain of p53
Antibodies to DNA are characteristic of many autoimmune diseases, notably systemic lupus erythematosus (SLE) and particularly lupus nephritis. However, there is at present no generally accepted explanation for the prevalence of anti-DNA antibodies in autoimmune disorders. Immunity to DNA appears to be driven by an antigen (Radic et al, 1994), but self-DNA is unlikely to be the driving antigen because mammalian DNA usually does not induce an anti-DNA immune response (Pisetsky, 1996).
It has been reported that immunization with monoclonal antibodies can induce immune responses that extend beyond the specificity of the antibody, probably by anti-idiotypic connectivity based on idiotypic-determinants in the variable regions of the immunizing monoclonal antibody.
According to idiotypic antibody network terminology, Ab1 is the first antibody, the antibody binding to the antigen, and Ab2 is the anti-idiotypic antibody to Ab1. The variable region of Ab2 may mimic the conformation of the antigen because both the antigen and Ab2 can be bound by Ab1. Ab3 is the anti-idiotypic antibody to Ab2. Because of the chain of structural complementarity, Ab1 and Ab3 can have similar specificity for the original antigen.
The PAb-421 antibody is a prototypic monoclonal antibody that reacts with the C-terminal DNA-binding domain of p53. The sequences of the variable heavy (VH) and variable light (VL) chains of the anti-p53 PAb-421 have been elucidated (see WO 98/56416). The use of PAb-421 antibody for the treatment of cancer was suggested, since it activated DNA binding of p53 in vitro (see WO 94/12202).
The inventors previously reported that immunization of mice with PAb-421 induced formation of anti-idiotypic antibodies that also bind DNA (Herkel et al., 2000; and WO 00/23082). Two of these monoclonal anti-idiotypic antibodies, designated Idi-1 and Idi-2, mimicked the binding properties of the p53 regulatory domain and reacted specifically with PAb-421 and double- and single stranded DNA.
It was suggested by the present inventors (after the priority date of the present invention) that damaged DNA has a chemically defined structure that is recognized by p53 and by Idi-1 and Idi-2 antibodies (Herkel, et al, 2004). Nowhere in the background art was it taught or suggested that it is possible to identify novel peptides having anti-apoptotic and anti-inflammatory properties using such anti-idiotypic antibodies.
There is an unmet need for novel compositions that may serve to attenuate cellular and immune stress-response in normal tissue, in a manner that is specific, safe and effective, thereby reducing the severity of stress associated degenerative diseases and stress-induced inflammation.